High-throughput structure determination using nucleic acid calipers

Patent metadata

Field	Value
Publication number	US-10914733-B2
Application number	US-201815862947-A
Country	US
Kind code	B2
Filing date	Jan 5, 2018
Priority date	Apr 23, 2014
Publication date	Feb 9, 2021
Grant date	Feb 9, 2021

Abstract

Provided herein are compositions and methods for determining the structure of individual targets using by determining long-range distances within such targets.

First claim

What is claimed is:

1. A system for performing structural analysis of a target bound to nucleic acid handles, comprising
a single-stranded nucleic acid caliper having a target domain comprising
(i) a nucleotide sequence TT1 that is complementary to nucleic acid handle TH1 bound to the target,
(ii) a target domain sequence comprising a looped structure when bound to the target, and
(iii) a nucleotide sequence TT2 that is complementary to nucleic acid handle TH2 bound to the target,

wherein the nucleic acid caliper is attached to a bead at a first end and is configured to be attached to, or is attached to, a solid surface at a second end.

2. The system of claim 1 , wherein the target domain is flanked by nucleotide sequence T1 at one end and nucleotide sequence T2 at the other end, and wherein T1 and T2 are complementary to a target splint.

3. The system of claim 2 , further comprising the target splint, wherein the target splint is a single-stranded oligonucleotide comprising
partial sequence complementarity to the single-stranded nucleic acid caliper at the nucleotide sequences TS1 and TS2, and
a TS toehold sequence that remains single-stranded when the target splint is bound to the single-stranded nucleic acid caliper.

4. The system of claim 1 , further comprising the target, wherein the target is bound to single-stranded nucleic acid handles, TH1 and TH2.

5. The system of claim 1 , wherein the nucleic acid caliper is attached to a solid surface at the second end.

6. The system of claim 1 , further comprising single stranded oligonucleotides comprising nucleic acid handle TH1 and single stranded oligonucleotides comprising nucleic acid handle TH2.

7. A method comprising
measuring, under tension, a bead-to-surface distance of a single-stranded nucleic acid caliper of claim 1 attached to a bead on a first end and to a surface on a second end, when bound to a target that is bound to single-stranded nucleic acid handles TH1 and TH2.

8. The method of claim 7 , wherein the target is a protein.

9. The method of claim 7 , wherein the target is a multi-unit protein.

10. The method of claim 8 , wherein the single-stranded nucleic acid handles, TH1 and TH2, are attached to the target at amino acids of same type.

11. The method of claim 8 , wherein the single-stranded nucleic acid handles, TH1 and TH2, are attached to the target at lysines.

12. The method of claim 8 , wherein the single-stranded nucleic acid handles, TH1 and TH2, are attached to the target at cysteines.

13. The method of claim 7 , wherein measuring is performed under non-denaturing conditions.

14. The method of claim 13 , wherein a second measuring step is performed under denaturing conditions.

15. The method of claim 7 , wherein under tension means a force of less than about 10 pN.

16. The method of claim 7 , wherein under tension means a force of about 300-1000 pN.

17. The method of claim 7 , wherein under tension means under centrifugal force or magnetic force.

18. The method of claim 1 , the nucleic acid caliper is bound to an Xaa residue and a Yaa residue of the target, wherein the Xaa and Yaa residues are attached to single stranded nucleic acid handles TH1 and TH2, respectively.

19. The method of claim 7 , wherein the method comprises
(a) measuring, under tension, the bead-to-surface distance of the nucleic acid caliper, when bound to an Xaa residue and a first Yaa residue of the target, wherein the Xaa and Yaa residues are attached to single stranded nucleic acid handles, TH1 and TH2,
(b) dissociating the nucleic acid caliper from the first Yaa residue and attaching the nucleic acid caliper to a second Yaa residue,
(c) measuring, under tension, the bead-to-surface distance of the nucleic acid caliper when bound to the Xaa and second Yaa residues of the target, and
(d) repeating steps (a)-(c).

20. The method of claim 18 , wherein steps (a) to (c) are performed multiple times at a force of less than about 10 pN, and then steps (a) to (c) are performed multiple times at a force of about 300-1000 pN.

Assignees

• Childrens Medical Center
• Dana Farber Cancer Inst Inc

Inventors

• Shih William M
• Wong Wesley Philip

Classifications

• C12Q1/68 Primary

  involving nucleic acids · CPC title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• G01N33/54333 Primary

  Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q2565/133

  conformational analysis · CPC title